Glucoamylase was produced extracellularly by fermentation of strain Aspergillus awamori, which had been genetically modified to have high-level glucoamylase activity. Initial experiments showed that the enzyme deactivated quickly, with a half-life of less than 6 days even stored at 5 degrees C. A possible reason for the rapid deactivation was the presence of proteases, attacking and degrading the glucoamylase. Therefore a liquid protease inhibitor cocktail (Sigma, USA) was selected and applied to enhance the stability of the enzyme. The activity of the enzyme (stored at 5 degrees C) measured by the Schoorl-method with starch as substrate showed that the cocktail was effective with the enzyme maintaining 95% of its initial storage activity for almost one year. The enzyme preparation has been used for starch hydrolysis in a flat-sheet membrane bioreactor at 60 degrees C to manufacture glucose solution and its operation stability extended by using the cocktail.

Candida rugosa lipase has shown to retain catalytic activity in ionic liquids. In this work, enantioselectivity of this enzyme in the esterification of 2-substituted-propanoic acids and 1-butanol is compared in ionic liquids and organic solvents. The role of solvent hydrophobicity (logP), water content and the effect of substituents are evaluated. Optimal water concentration in the reaction media was determined, where the enzyme shows maximal activity and enantioselectivity. Enantioselectivity can be improved when chlorine substituent was replaced by slightly bigger size bromine. Contrary to reactions in common organic solvents, there was no need for purification steps following the reaction in ionic liquids in order to recycle the enzyme. In 1-butyl-3-methyl-imidazolium-hexafluoro-phosphate ([bmim]PF6) and 1-octyl-3-nonyl-imidazolium-hexafluoro-phosphate ([onim]PF6) ionic liquids, C. rugosa lipase could be recycled five times without appreciable activity or enantioselectivity losses.

The application of ionic liquids as solvents for the enantioselective esterification of (R,S)-2-chloropropanoic acid with butan-1-ol using Candida rugosa lipase is reported. The role of water produced during the reaction and controlling of the water activity with pervaporation was studied. The enantioselective esterification of (R, S)-2-chloropropanoic acid with butan-1-ol in different organic solvents and ionic liquids was studied. The reaction was effectively catalysed by Candida rugosa lipase. From the ionic solvents investigated, the best results were achieved in [bmim]PF6. The presence of water had a strong effect on the activity of the lipase. Since in esterification reactions with acid and alcohol equimolar amounts of water are formed, the excess of water was removed and the water activity was kept constant using a pervaporation system without any additives. At the optimum water activity Candida rugosa lipase showed high thermal stability in [bmim]PF6 and it could be reused for at least five recycles with only a small lost of activity.